Monoclonal antibodies against human colon carcinoma-associated antigens and uses therefor

ABSTRACT

Monoclonal antibodies, in particular 33.28 and 31.1, and chimeric antibodies, in particular mouse/humans chimeric Chi #1 specific for glycoprotein antigens of colon carcinoma-associated antigens which are immunogenic in humans, are disclosed. Such antibodies, and fragments and derivatives thereof, are useful in immunodiagnosis and immunotherapy of human colon, breast, and ovarian cancer, and for purification of antigens which can serve as immunotherapeutic agents. Methods of detecting the colon carcinoma-associated antigen in a sample, and methods for treating subjects having colon, breast, and ovarian carcinomas are disclosed.

This application is a continuation-in-part of U.S. application Ser. No.08/159,836 filed Nov. 30, 1993, which is a continuation-in-part of U.S.application Ser. No. 08/117,430, filed Sep. 7, 1993, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 07/670,816,filed Mar. 18, 1991, now abandoned, which is a continuation-in-part ofU.S. application Ser. No. 07/176,337, filed Mar. 31, 1988, nowabandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention, in the field of immunology and medicine, relates to newhybridoma lines and the monoclonal antibodies (mAbs) they secrete whichare specific for clinically defined colon carcinoma-associated antigens.The mAbs are useful in vivo for immunodetection and immunotherapy ofcolon carcinoma as well as for the detection and purification of coloncarcinoma-associated antigens.

2. Description of the Background Art

During the process of oncogenesis, a number of cell-surface molecules ormarkers appear on cells. Such tumor-related markers includeoncofetoproteins, neoglycoproteins, sphignolipids, and modifications ofexisting surface proteins. Such new or altered structures are often shedfrom the tumor cell surface and appear in the serum or in otherbiological fluids. The detection of any of these substances or “tumormarkers” serves as the basis for diagnosing or monitoring the progressof neoplastic disease.

Early animal studies demonstrated that, among these tumor markers, asubset of tumor membrane protein or glycoprotein antigens wereimmunogenic. Upon appropriate reintroduction into the tumor-bearinghost, typically after surgical removal of the primary tumor, suchantigens could effectively block the establishment of new tumor growth.

An attempt to use similarly derived tumor-associated antigens in humanswas made by Hollinshead and Stewart using a relatively purified membranepreparation in patients with lung cancer (Stewart, T. H. M. et al., Ann.N.Y. Acad. Sci. 277:436 (1976)). These studies were later expanded toinclude patients with melanoma and colon carcinoma, wherein differentpooled allogeneic tumor preparations were administered in combinationwith complete Freund's adjuvant (Hollinshead, A. C. et al., Cancer4:9:1387 (1982); Hollinshead, A. C. et al., Cancer 56:480 (1985)).

The use of Freund's adjuvant was based on observations that normaltissue antigens with this adjuvant produced severe autoimmune responsesin animal recipients, whereas in the absence of this adjuvant, noadverse reactions were seen. The adjuvant was thought to promote antigenprocessing by host macrophages as well as prolong the stimulatory actionof the antigen at the site of its deposition (see, for example, Roitt,L. Essential Immunology, 6th Ed., Blackwell Scientific Publications,Oxford (1988)).

The above observations served as the basis for early clinical trialsusing specific human tumor membrane proteins and glycoproteins as tumor“vaccines.” Various of the tumor-associated antigens which had beenisolated and characterized could prolong survival and, in some cases,produce regression of metastatic disease.

With the advent of monoclonal antibody (mAb) technology, it has becomepossible to obtain pure antibody populations which permit betterpurification and characterization of the various tumor markers andtumor-associated antigens that are useful for immunodiagnosis orimmunotherapy. Many mAbs have been described that have varying degreesof selectivity for tumor antigens (versus normal cell surface markers);some of these tumor antigens are broadly represented across several ormany tumor types, whereas others appear to be truly tumor-specific. Anumber of these mAb-tumor antigen systems are described below.

Herlyn et al., Proc. Natl. Acad. Sci. USA 76:1438 (1979), discloses twomAbs obtained by immunizing mice with human colorectal carcinoma (CRC)cells. The mAbs have selective reactivity with human CRC cells. One mAb,1083-17 (the forerunner of 17-1A), is now known to react with a 41 kDaglycoprotein (see below).

Herlyn et al., J. Clin. Immunol. 2:135 (1982), described the detectionof a circulating colorectal carcinoma (CRC)-associated antigen by a mAbdeveloped against a membrane antigen of the SW116 cell line. MAbs 19-9and 52a, which recognize a monosialoganglioside antigen (Magnani, J. L.et al., Science 212:55 (1981)), reacted with cells of 8 of 12 CRC linesas well as with the cells of one gastric carcinoma and one pancreaticcarcinoma. MAb C₄14 reacted with four of six CRC cultures and withgastric tumor cells. The binding of mAbs 19-9 and 52a to tumor cellswere inhibited by a CRC patient's serum. However, CRC sera inhibitedbinding less frequently than did sera from patients with pancreatic orgastric tumors.

Girardet et al., J. Immunol. 136:1497-1503 (1986), disclosed mAbsagainst human colon carcinomas. The L-D1 mAb reacted with a 41 kDaglycoprotein, believed to be the same antigen as that defined by mAb1083-17-1A (Herlyn et al., 1979, supra). The L-C5 mAb precipitatedproteins having molecular weights of 43, 45, 47 and 53 kDa from LoVocolon carcinoma cells. L-D1 did react with cervical carcinoma lines,while L-C5 reacted with breast carcinoma lines. Their binding topancreatic carcinomas was not examined.

Greiner et al., Science 235:895-898 (1987), discloses mAb 06.2 whichreacts with a 90 kDa glycoprotein allegedly found in 75-80% of breastcarcinomas and more than 90% of colon carcinomas.

Sakamoto et al., U.S. Pat. No. 4,579,827 (Apr. 1, 1986), discloses anumber of mAbs said to be useful for diagnosing or treating human coloncancer by a number of different approaches. None of these mAbs is shownto react with a human colon carcinoma-associated antigen that is aprotein of either 61 or 72 kDa molecular weight, distinguishing theseantibodies from the antibodies 33.28 antibody of the present invention(described below). Furthermore, none of the Sakamoto mAbs have thedegree of colon tumor specificity of the mAbs disclosed in the presentapplication.

Delaloye et. al., J. Clin. Invest. 77:301 (1986), discloses the use of amAb specific for carcinoembryonic antigen (CEA) to detect colorectalcarcinoma in vivo using ¹²³I-labelled fragments and emissioncomputerized tomography. The mAb described bears no relation to the mAbsof the present invention.

Douillard et al., Hybridoma 5, Suppl. 1:S139 (1986), describes mAb 17-1Aand its cytotoxic properties to gastrointestinal adenocarcinomas invitro. 17-1A was used with some degree of success in immunotherapytrials. This mAb is said to recognize a 38-41 kDa protein and has abroad range of reactivity and lack of colon tumor specificity, clearlydistinguishing it from the antibodies of the present invention.

Scannon et al., U.S. Pat. No. 4,590,071 (May 20, 1986), discloses mAbsspecific for melanoma antigens conjugated to toxic proteins such asricin A chain and the use of these compositions to treat melanoma. Thereis no disclosure directed to colon tumor antigens or antibodies andtheir uses.

The relatively pure antigen preparation containing the immunogenic coloncarcinoma membrane antigens to which the mAbs of the present inventionare directed was originally described by Hollinshead et al., Science177:887-889 (1972).

The clinical evaluation of the above antigen preparation, including adescription of its immunogenicity and potential for enhancing patientsurvival through stimulation of specific active immunity, was describedby Hollinshead et al., 1985, supra.

The work of the present inventors leading to the present invention isbriefly described in an abstract by Tsang et al., “Monoclonal Antibodiesto Human Colon Carcinoma Associated Antigens,” Intl. Symp. Biotech. inClin. Med., Rome, Italy, Apr. 13-15, 1987. This reference was madeavailable to the public less than one year before the filing of theultimate parent application (U.S. Ser. No. 07/176,337) for the presentapplication.

SUMMARY OF THE INVENTION

The present inventors have produced murine mAbs and mouse-human chimericantibodies specific for colon carcinoma-associated antigens which wereknown to be immunogenic in humans. These antigens, isolated in theinventors' laboratory, are unique among the previously described coloncancer antigens in that (1) the epitopes recognized by the mAbs are ofthe protein and not the carbohydrate component of tumor-associatedglycoproteins; (2) the antigens are not expressed in normal tissues; (3)the antigens are tumor-specific, being present in malignancies of colon,breast, and ovarian cancer; (4) the antigens are immunogenic in humans,having the capability of enhancing host anti-tumor immunity by bothcellular as well as humoral responses, thus improving survival in cancerpatients; and (5) the immunogenicity in humans is specific, in that onlycolon, breast and ovarian cancer patients, but not patients with otherforms of cancer, show evidence of specific in vivo or in vitroimmunological reactivity to the antigens.

The mAbs and chimeric antibodies of the present invention are useful fordiagnosis or therapy of colon, breast, and ovarian carcinoma, forexample by imaging metastatic tumors, by delivering cytotoxic agents tothe tumors, and by activating host effector mechanisms such asantibody-dependent cellular cytotoxicity (ADCC) or complement dependentcytotoxicity (CDC) to directly kill tumor cells.

The present invention is thus directed to a monoclonal antibody specificfor a human colon carcinoma-associated protein antigen wherein theantigen is specifically immunogenic in humans, and the antigen is notdetectable on normal human tissues or on human carcinoma cells otherthan colon, breast and ovarian carcinoma cells .

The present invention is also directed to a chimeric antibody specificfor a human colon carcinoma-associated protein antigen wherein theantigen is not detectable on normal human tissues or on human carcinomacells other than colon carcinoma cells . Mouse hybridoma PCA 31.1 hasbeen deposited at ATCC on Sep. 22, 2000 and assigned ATCC HB-12314Accession No. PTA-2497. Mouse hybridoma PCA 33.28 has been deposited atATCC on Aug. 26, 2003 and assigned ATCC HB-12315 Accession No. PTA-5413.Cells transfected with chimeric 31.1 have been deposited at ATCC on Mar.13, 1997 and assigned ATCC Accession No. CRL-12316. The above depositswere made are maintained at American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20862 USA on Mar. 13, 1997 10801University Boulevard, Manassas, Va. 20110-2209.

In one embodiment, the antibody is specific for a CCAA which is aprotein having a molecular weight of about 61 kilodaltons. In anotherembodiment, the antibody is specific for a CCAA which is a proteinhaving a molecular weight of about 72 kilodaltons. In a preferredembodiment, the antibody is the mouse monoclonal antibody 33.28 or 31.1or an antibody which binds specifically to the same coloncarcinoma-associated epitope as that bound by 33.28 or 31.1. In anotherpreferred embodiment, the antibody is a mouse/human chimeric antibodyChi #1 that binds specifically to the same colon carcinoma-associatedepitope as that bound by 31.1.

The present invention is also directed to the above antibody immobilizedon a solid phase.

The present invention includes the above antibody delectably labelled,for example, with a radiolabel.

In additional embodiments, the above antibody is conjugated to acytotoxic radionuclide, a cytotoxic drug, or a cytotoxic protein.

In yet another embodiment, the present invention is directed tomonoclonal antibodies against the above antibodies, i.e., secondgeneration monoclonal antibodies.

In a further embodiment, the present invention is directed to thirdgeneration monoclonal antibodies, i.e., monoclonal antibodies directedagainst the above second generation monoclonal antibodies.

The present invention is also directed to the above-discussed coloncarcinoma-associated antigens which are unique in that (1) the epitopesrecognized by the mAbs are of the protein and not the carbohydratecomponent of tumor-associated glycoproteins; (2) the antigens are notexpressed in normal tissues; (3) the antigens are tumor-specific, beingpresent in the malignancies of colon, breast and ovarian cancer; (4) theantigens are immunogenic in humans, having the capability of enhancinghost anti-tumor immunity, thus improving survival in cancer patients;and (5) the immunogenicity in humans is specific, in that only colon,breast and ovarian cancer patients, but not patients with other forms ofcancer, show evidence of specific in vivo or in vitro immunologicalreactivity to the antigens.

To date, all other purified antigens that have been used have failed toelicit both a cellular and a humoral response.

The present invention also provides a pharmaceutical composition usefulfor the immunotherapy of colon, breast and ovarian cancer comprising anantibody, fragment or derivative, as above, conjugated to a cytotoxicradionuclide, a cytotoxic drug, or a cytotoxic protein, in a suitableexcipient.

The present invention includes an immunoassay method for detecting in asample a colon carcinoma-associated antigen capable of binding to the33.28 or 31.1 murine monoclonal antibody or Chi #1, comprising:

-   -   (a) contacting the sample with an antibody described above; and    -   (b) detecting the antigen by detecting the binding of the        antibody.

In another embodiment, the invention provides as imaging method fordetecting a colon carcinoma-associated antigen in a subject, comprising:

-   -   (a) contacting the detectably labelled antibody as described        above with the subject; and    -   (b) detecting the antigen.

The present invention also includes a method of killing cells carrying acolon carcinoma-associated antigen, comprising:

-   -   (a) delivering to the cells an antibody as above, and a        cytotoxic effector agent; and    -   (b) allowing the killing to occur.        The effector agent may be complement, or effector cells active        in ADCC. Alternatively, antibodies labelled conjugated with a        cytotoxic radionuclide, drug or protein may be used directly.

The present invention is further directed to a method of treating asubject suspected of having a colon, breast and ovarian carcinomabearing an antigen which is capable of binding to the 33.28 or 31.1monoclonal antibody, or Chi #1 chimeric antibody comprisingadministering to the subject an effective dose of a pharmaceuticalcomposition as described above.

Also provided is a method for producing an immunogenic compositionuseful for clinical immunotherapy of colon, breast, and ovariancarcinoma, comprising:

-   -   (a) preparing a membrane extract of a tumor or cell line bearing        an antigen which is capable of binding to the 33.28 or 31.1        monoclonal antibody or Chi #1 antibody; and    -   (b) isolating the antigen by affinity purification using an        antibody as described above,        thereby producing the immunogenic composition.

In another embodiment, the present invention is directed to the use ofthe above antigen to produce a vaccine.

The present invention also includes a method of detecting and diagnosingcolon, breast and ovarian cancer by staining monoclonal antibody orchimeric antibody bound to the above-described human coloncarcinoma-associated antigen.

In another embodiment, the present invention includes a kit forselectively characterizing colon, breast, and ovarian carcinomas.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a tracing showing an HPLC elution profile of the Hollinshead“vaccine,” a partially purified preparation of colon carcinoma cellmembranes.

FIG. 2 is a tracing showing an HPLC elution profile of the coloncarcinoma-associated antigen obtained by affinity purification of thematerial contained in peak 4 of FIG. 1.

FIG. 3 is a graph showing the biodistribution of mAb 31.1 in nude micebearing a xenografted human tumor, LS-174T.

FIG. 4 is a graph showing the biodistribution of mAb 33.28 in nude micebearing a xenografted human tumor, LS-174T.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides antibodies, including monoclonal andchimeric antibodies, that are specific for, and capable of binding to,immunogenic human colon carcinoma-associated antigens (CCAA) which areprotein in nature. These antibodies are useful for diagnostic andtherapeutic purposes in subjects having or developing colon, breast orovarian carcinoma.

The present invention provides not only mouse mAbs, but also chimericantibodies which are constructed from mouse V regions derived from themAbs of the present invention. Thus, the chimeric antibodies maintainthe ability to recognize the same CCAA epitopes as the mAbs.

The term “epitope” refers to that portion of any molecule capable ofbeing recognized by, and bound by, an antibody. In general, epitopesconsist of chemically active surface groupings of molecules, forexample, amino acids or sugar side chains, and have specificthree-dimensional structural characteristics as well as specific chargecharacteristics. The epitopes of interest for the present invention areepitopes comprising amino acids.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce an antibody capable of binding to an epitope of that antigen.An antigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

The term “antibody” is meant to include both intact immunoglobulinmolecules as well as fragments and derivatives thereof, such as, forexample, Fab, Fab′, F(ab′)₂ and Fv, which are capable of bindingantigen. These fragments lack the Fc fragment of intact antibody, clearmore rapidly from the circulation, and may have less non-specific tissuebinding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325(1983)). These fragments are produced from intact antibodies usingmethods well known in the art, for example by proteolytic cleavage withenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)₂ fragments).

A “derivative” of an antibody contains additional chemical moieties notnormally a part of the protein. Covalent modifications of the proteinare included within the scope of this invention. Such modifications maybe introduced into the molecule by reacting targeted amino acid residuesof the antibody with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. For example,derivatization with bifunctional agents, well-known in the art, isuseful for cross-linking the antibody or fragment to a water-insolublesupport matrix or to other macromolecular carriers.

By “vaccine” is meant an agent used to stimulate the immune system of aliving organism so that immunological protection against further harmcaused by an infectious agent is provided. Administration of a vaccinecontemplated by the present invention to the patient may be by any knownor standard techniques. These include oral ingestion, intestinalintubation, or broncho-nasal spraying. Other methods of administration,such as intravenous injection, that allow the carrier microbe to reachthe human or animal's bloodstream may be acceptable when the carriermicrobe is unable to reproduce.

The antibodies of the present invention are novel in that they are thefirst known mAbs and chimeric antibody specific for CCAA wherein thetumor antigens are known to be immunogenic in humans. That is, theantigens recognized by the antibodies of the present invention induce animmune response in patients with colon, breast, and ovarian cancer, butnot in other individuals, such as patients with other types of cancer.The immunogenicity of these antigens is expressed chiefly ascell-mediated immunity, measurable either by assay of delayed cutaneoushypersensitivity in vivo (“skin tests”), or by various in vitro assaysof specific lymphocyte reactivity, such as lymphocyte proliferation orlymphocyte migration inhibition assays. For general principles ofimmunogenicity and description of various assays of specificimmunological reactivity, see: Roitt, I., Essential Immunology, 6th Ed.,Blackwell Scientific Publications, Oxford (1988); Roitt, I. et al.,Immunology, C. V. Mosby Co., St. Louis, Mo. (1985); Klein, J.,Immunology, Blackwell Scientific Publications, Inc., Cambridge, Mass.(1990); Klein, J., Immunology: The Science of Self-NonselfDiscrimination, John Wiley & Sons, New York, N.Y. (1982); Paterson, P.Y., Textbook of Immunopathology, Grune and Stratton, New York, (1986),which are hereby incorporated by reference.

In a preferred embodiment, the antibody of the present invention is amurine mAb designated 33.28. In another preferred embodiment, theantibody is a murine mAb designated 31.1. In yet another embodiment theantibody is a chimeric antibody which recognizes an epitope recognizedby 33.28. In another embedment, the antibody is a chimeric antibodywhich recognizes an epitope recognized by 31.1.

The chimeric antibodies of the invention comprise individual chimericheavy (H) and light (L) immunoglobulin chains. The chimeric H chaincomprises an antigen-binding region derived from the H chain of anon-human antibody specific for the epitope recognized by 33.28 or 31.1,which is linked to at least a portion of a human H chain C region(C_(H)).

A preferred chimeric L chain comprises an antigen-binding region derivedfrom the L chain of either the 33.28 or 31.1 mAb, linked to at least aportion of a human L chain C region (C_(L)).

Alternatively, a preferred chimeric H chain comprises an antigen-bindingregion derived from the L chain of either the 33.28 or 31.1 mAb, linkedto at least a portion of a human L chain C region (C_(H)).

As used herein, the term “antigen-binding region” refers to that portionof an antibody molecule which contains the amino acid residues thatinteract with an antigen and confer on the antibody its specificity andaffinity for the antigen. The antibody region includes the “framework”amino acid residues necessary to maintain the proper conformation of theantigen-binding residues.

As used herein, the term “chimeric antibody” includes monovalent,divalent or polyvalent immunoglobulins. A monovalent chimeric antibodyis a dimer (HL) formed by a chimeric H chain associated throughdisulfide bridges with a chimeric L chain. A divalent chimeric antibodyis tetramer (H₂L₂) formed by two HL dimers associated through at leastone disulfide bridge. A polyvalent chimeric antibody can also beproduced, for example, by employing a C_(H) region that aggregates(e.g., from an IgM H chain, or μ chain).

The invention also provides for “derivatives” of the monoclonal orchimeric antibodies, which term includes those proteins encoded bytruncated or modified genes to yield molecular species functionallyresembling the immunoglobulin fragments. The modifications include, butare not limited to, addition of genetic sequences coding of cytotoxicproteins such as plant and bacterial toxins. The fragments andderivatives can be produced from prokaryotic or eukaryotic hosts, asdescribed herein by recombinant means. Alternatively, the fragments andderivatives may be produced by chemical means, such as proteolyticcleavage of intact immunoglobulin molecules, or other chemicalmodifications or derivatizations known in the art. Such derivatizedmoieties may improve the solubility, absorption, biological half-life,and the like. The moieties may alternatively eliminate or attenuate anyundesirable side effect of the antibody protein. Moieties capable ofmediating such effects are disclosed, for example, in Remington'sPharmaceutical Sciences, 16th ed., Hack Publishing Co., Easton, Pa.(1980).

Antibodies, fragments or derivatives having chimeric H chains and Lchains of the same or different V region binding specificity can beprepared by appropriate association of the individual polypeptidechains, as taught, for example by Sears et al., Proc. Natl. Acad. Sci.USA 72:353∝357 (1975). With this approach, hosts expressing chimeric Hchains (or their derivatives) are separately cultured from hostsexpressing chimeric L chains (or their derivatives) and theimmunoglobulin chains are separately recovered and then associated.Alternatively, the hosts can be co-cultured and the chains allowed toassociate spontaneously in the culture medium, followed by recovery ofthe assembled immunoglobulin, fragment or derivative.

Murine hybridomas which produce mAb specific for CCAA, such as the 33.28and 31.1 mAbs of the present invention, are formed by the fusion of amouse fusion partner cell, such as SP2/0, and spleen cells from miceimmunized against the CCAA.

Mice may be immunized with crude or semi-purified preparationscontaining the antigens of interest, such as, for example, theHollinshead “vaccine,” which is a partially purified membranepreparation of colon-carcinoma cells (Hollinshead et al., supra). Toimmunize the mice, a variety of different conventional protocols may befollowed. For example, mice may receive primary and boostingimmunizations of antigenic preparations.

The cell fusions are accomplished by standard procedures well known tothose skilled in the field of immunology (Kohler and Milstein, Nature256:495-497 (1975) and U.S. Pat. No. 4,376,110; Hartlow, E. et al.,supra; Campbell, A., “Monoclonal Antibody Technology,” In: LaboratoryTechniques in Biochemistry and Molecular Biology, Volume 13 (Burdon, R.,et al., eds. ), Elsevier, Amsterdam (1984); Kennett et al., MonoclonalAntibodies (Kennett et al., eds. pp. 365-367, Plenum Press, N.Y., 1980);de St. Groth, S. F., et al., J. Immunol. Meth. 35:1-21 (1980); Galfre,G. et al., Methods Enzymol. 73:3-46 (1981); Goding, J. W. 1987,Monoclonal Antibodies: Principles and Practice, 2nd ed. Academic Press,London, 1987).

Fusion partner cell lines and methods for fusing and selectinghybridomas and screening for mAbs are well known in the art (Hartlow, E.et al., supra; Kawamoto, T. et al., Meth. Enzymol. 121:266-277 (1986);Kearney, J. F. et al., J. Immunol. 123:1548-1550 (1979); Kilmartin, J.V. et al., J. Cell Biol. 93:576-582 (1982); Kohler, G. et al., Eur. J.Immunol. 6:292-295 (1976); Lane, D. P. et al., J. Immunol. Meth.47:303-307 (1981); Mueller, U. W. et al., J. Immunol. Meth. 87:193-196(1986); Pontecorvo, G., Somatic Cell Gener. 1:397-400 (1975); Sharo, J.,et al., Proc. Natl. Acad. Sci. USA 7:6:1420-1424 (1979); Shulman, M. etal., Nature 276:269-270 (1978); Springer, T. A. (ed), HybridomaTechnology in the Biosciences and Medicine, Plenum Press, New York,1985; and Taggart, R. T. et al., Science 219:1228-1230 (1982)).

The mAbs of the present invention may be produced in large quantities byinjecting hybridoma cells secreting the antibody into the peritonealcavity of mice and, after appropriate time, harvesting the ascites fluidwhich contains a high titer of the mAb, and isolating the mAb therefrom.Alternatively, the mAbs may be produced by culturing hybridoma cells invitro and isolating the secreted mAb from the cell culture medium.

Human genes which encode the C regions of the chimeric antibodies of thepresent invention are derived from cells which express, and preferably,produce, human immunoglobulins. The human C_(H) region can be derivedfrom any of the known classes or isotypes of human H chains, includinggamma, μ, α, δ or ε. Since the H chain isotype is responsible for thevarious effector functions of an antibody, the choice of C_(H) regionwill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity(ADCC). Preferably, the C_(H) region is derived from gamma 1 (IgG1),gamma 3 (IgG3), gamma 4 (IgG4), and μ (IgM).

The human C_(L) region can be derived from either human L chain isotype,kappa or lambda.

Genes encoding human immunoglobulin C regions are obtained from humancells by standard cloning techniques (Sambrook, J. et al., MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1989)). Human C region genes are readilyavailable from known clones containing genes representing the twoclasses of L chains and the five classes of H chains. Chimeric antibodyfragments, such as F(ab′)₂ and Fab, can be prepared by designing achimeric H chain gene which is appropriately truncated. For example, achimeric gene encoding the H chain portion of an F(ab′)₂ fragment wouldinclude DNA sequences encoding the CH₁ domain and hinge region of the Hchain, followed by a translational stop codon to yield the truncatedmolecule.

Generally, the chimeric antibodies of the present invention are producedby cloning DNA segments encoding the H and L chain antigen-bindingregions of a CCAA-specific antibody, preferably non-human, mostpreferably 33.28 or 31.1, and joining these DNA segments to DNA segmentsencoding human C_(H) and C_(L)regions to produce chimericimmunoglobulin-encoding genes.

Thus, in a preferred embodiment, a fused gene is created which comprisesa first DNA segment that encodes at least the antigen-binding region ofnon-human origin, such as a functionally rearranged V region withjoining (J) segment, linked to a second DNA segment encoding at least apart of a human C region. This fusion can be accomplished by thepolymerase chain reaction, as reported by Fernando et al., Miami Symp.Short Reports 3: 88, 1993.

The DNA encoding the antibody-binding region may be genomic DNA or cDNA.A convenient alternative to the use of chromosomal gene fragments as thesource of DNA encoding the murine V region antigen-binding segment isthe use of cDNA for the construction of chimeric immunoglobulin genes,as reported by Liu et al., Proc. Natl. Acad. Sci., USA 84:3439 (1987)and J. Immunology 139:3521 (1987), which references are herebyincorporated by reference. The use of cDNA requires that gene expressionelements appropriate for the host cell be combined with the gene inorder to achieve synthesis of the desired protein. The use of cDNAsequences is advantageous over genomic sequences (which containintrons), in that cDNA sequences can be expressed in bacteria or otherhosts which lack appropriate RNA-splicing systems.

Therefore, in an embodiment utilizing cDNA encoding the antibody Vregion, the method of producing the chimeric antibody involves severalsteps, outlined below:

-   -   1. Isolation of messenger RNA (mRNA) from the cell line        producing the monoclonal antibody, cloning and cDNA production        therefrom;    -   2. Preparation of a full length cDNA library from purified mRNA        from which the appropriate V region gene segments of the L and H        chain genes can be: (i) identified with appropriate probes, (ii)        sequenced, and (iii) made compatible with a C gene segment;    -   3. Preparation of C region gene segments by cDNA preparation and        cloning;    -   4. Construction of complete H or L chain coding sequences by        linkage of the cloned specific V region gene segments to cloned        human C region gene, as described above;    -   5. Expression and production of chimeric L and H chains in        selected hosts, including prokaryotic and eukaryotic cells.

One common feature of all immunoglobulin H and L chain genes and theirencoded mRNAs is the J region. H and L chain J regions have differentsequences, but a high degree of sequence homology exists (greater than80%) among each group, especially near the C region. This homology isexploited in this method and consensus sequences of H and L chain Jregions may be used to design oligonucleotides for use as primers forintroducing useful restriction sites into the J region for subsequentlinkage of V region segments to human C region segments.

C region cDNA vectors prepared from human cells can be modified bysite-directed mutagenesis to place a restriction site at the analogousposition in the human sequence. For example, one can clone the completehuman kappa chain C (C_(k)) region and the complete human gamma-1 Cregion (C_(gamma-1)). In this case, the alternative method based upongenomic C region clones as the source for C region vectors would notallow these genes to be expressed in bacterial systems where enzymesneeded to remove intervening sequences are absent. Cloned V regionsegments are excised and ligated to L or H chain C region vectors.Alternatively, the human C_(gamma-1) region can be modified byintroducing a termination codon thereby generating a gene sequence whichencodes the H chain portion of an Fab molecule. The coding sequenceswith linked V and C regions are then transferred into appropriateexpression vehicles for expression in appropriate hosts, prokaryotic oreukaryotic.

Two coding DNA sequences are said to be “operably linked” if the linkageresults in a continuously translatable sequence without alteration orinterruption of the triplet reading frame. A DNA coding sequence isoperably linked to a gene expression element if the linkage results inthe proper function of that gene expression element to result inexpression of the coding sequence.

Expression vehicles include plasmids or other vectors. Preferred amongthese are vehicles carrying a functionally complete human C_(H) or C_(L)chain sequence having appropriate restriction sites engineered so thatany V_(H) or V_(L) chain sequence with appropriate cohesive ends can beeasily inserted therein. Human C_(H) or C_(L) chain sequence-containingvehicles thus serve as intermediates for the expression of any desiredcomplete H or L chain in any appropriate host.

A chimeric mouse-human antibody will typically be synthesized from genesdriven by the chromosomal gene promoters native to the mouse H and Lchain V regions used in the constructs; splicing usually occurs betweenthe splice donor site in the mouse J region and the splice acceptor sitepreceding the human C region and also at the splice regions that occurwithin the human C_(H) region; polyadenylation and transcriptiontermination occur at native chromosomal sites downstream of the humancoding regions.

Gene expression elements useful for the expression of cDNA genesinclude: (a) viral transcription promoters and their enhancer elements,such as the SV40 early promoter (Okayama, H. et al., Mol. Cell. Biol.3:280 (1983)), Rous sarcoma virus LTR (Gorman, C. et al., Proc. Natl.Acad. Sci., USA 79:6777 (1982)), and Moloney murine leukemia virus LTR(Grosschedl, R. et al., Cell 41:885 (1985)); (b) splice regions andpolyadenylation sites such as those derived from the SV40 late region(Okayama et al., supra); and (c) polyadenylation sites such as in SV40(Okayama et al., supra).

Immunoglobulin cDNA genes may be expressed as described by Liu et al.,Supra, and Weidle et al., Gene 51:21 (1987), using as expressionelements the SV40 early promoter and its enhancer, the mouseimmunoglobulin H chain promoter enhancers, SV40 late region mRNAsplicing, rabbit β-globin intervening sequence, immunoglobulin andrabbit β-globin polyadenylation sites, and SV40 polyadenylationelements. For immunoglobulin genes comprised of part cDNA, part genomicDNA (Whittle et al., Protein Engineering 1:499 (1987)), thetranscriptional promoter may be human cytomegalovirus (CMV), thepromoter enchancers derived from CMV and mouse/human immunoglobulin, andmRNA splicing and polyadenylation regions derived from the nativechromosomal immunoglobulin sequences.

In one embodiment, for expression of cDNA genes in rodent cells, thetranscriptional promoter is a viral LTR sequence, the transcriptionalpromoter enchancers are either or both the mouse immunoglobulin heavychain enhancer and the viral LTR enhancer, the splice region contains anintron of greater than 31 bp, and the polyadenylation and transcriptiontermination regions are derived from the native chromosomal sequencecorresponding to the immunoglobulin chain being synthesized. In otherembodiments, cDNA sequences encoding other proteins are combined withthe above-recited expression elements to achieve expression of theproteins in mammalian cells.

Each fused gene is assembled in, or inserted into, an expression vector.Recipient cells capable of expressing the chimeric immunoglobulin chaingene product are then transfected singly with a chimeric H or chimeric Lchain-encoding gene, or are co-transfected with a chimeric H and achimeric L chain gene. The transfected recipient cells are culturedunder conditions that permit expression of the incorporated genes andthe expressed immunoglobulin chains or intact antibodies or fragmentsare recovered from the culture. In one embodiment, the fused genesencoding the chimeric H and L chains, or portions thereof, are assembledin separate expression vectors that are then used to co-transfect arecipient cell.

Each vector may contain two selectable genes, a first selectable genedesigned for selection in a bacterial system and a second selectablegene designed for selection in a eukaryotic system, wherein each vectorhas a different pair of genes. This strategy results in vectors whichfirst direct the production, and permit amplification, of the fusedgenes in a bacterial system. Subsequently, the genes so produced andamplified in a bacterial host are subsequently used to co-transfect aeukaryotic cell, and allow selection of a co-transfected cell carryingthe desired transfected genes.

Examples of selectable genes of use in a bacterial system are the genethat confers resistance to ampicillin and the gene that confersresistance to chloramphenicol. Preferred selectable genes for use ineukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (designated gpt) and the phosphotransferase gene fromTn5 (designated neo). Selection of cells expressing gpt is based on thefact that the enzyme encoded by this gene utilizes xanthine as asubstrate for purine, nucleotide synthesis, whereas the analogousendogenous enzyme cannot. In a medium containing (1) mycophenolic acid,which blocks the conversion of inosine monophosphate to xanthinemonophosphate, and (2) xanthine, only cells expressing the E gene cansurvive. The product of the neo blocks the inhibition of proteinsynthesis by the antibiotic G418 and other antibiotics of the neomycinclass.

The two selection procedures can be used simultaneously or sequentiallyto select for the expression of immunoglobulin chain genes introduced ontwo different DNA vectors into a eukaryotic cell. It is not necessary toinclude different selectable markers for eukaryotic cells; an H and an Lchain vector, each containing the same selectable marker can beco-transfected. After selection of the appropriately resistant cells,the majority of the clones will contain integrated copies of both H andL chain vectors.

Alternatively, the fused genes encoding the chimeric H and L chains canbe assembled on the same expression vector.

For transfection of the expression vectors and production of thechimeric antibody, the preferred recipient cell line is a myeloma cell.Myeloma cells can synthesize, assemble and secrete immunoglobulinsencoded by transfected immunoglobulin genes and possess the mechanism orglycosylation of the immunoglobulin. A particularly preferred recipientcell is the Ig-non-producing myeloma cell SP2/0 (ATCC #CRL 8287). SP2/0cells produce only immunoglobulin encoded by the transfected genes.Myeloma cells can be grown in culture or in the peritoneal cavity of amouse, where secreted immunoglobulin can be obtained from ascites fluid.Other suitable recipient cells include lymphoid cells such as Blymphocytes of human or non-human origin, hybridoma cells of human ornon-human origin, or interspecies heterohybridoma cells.

The expression vector carrying a chimeric antibody construct of thepresent invention may be introduced into an appropriate host cell by anyof a variety of suitable means, including such biochemical means astransformation, transfection, conjugation, protoplast fusion, calciumphosphate-precipitation, and application with polycations such asdiethylaminoethyl (DEAE) dextran, and such mechanical means aselectroporation, direct microinjection, and microprojectile bombardment(Johnston et al., Science 240:1538 (1988)). A preferred way ofintroducing DNA into lymphoid cells is by electroporation (Potter etal., Proc. Natl. Acad. Sci. USA 81:7161 (1984); Yoshikawa, K. et al.,Jpn. J. Cancer Res. 77:1122-1133). In this procedure, recipient cellsare subjected to an electric pulse in the presence of the DNA to beincorporated. Typically, after transfection, cells are allowed torecover in complete medium for about 24 hours, and are then seeded in96-well culture plates in the presence of the selective medium. G418selection is performed using about 0.4 to 0.8 mg/ml G418. Mycophenolicacid selection utilizes about 6 μg/ml plus about 0.25 mg/ml xanthine.The electroporation technique is expected to yield transfectionfrequencies of about 10⁻⁵ to about 10⁻⁴ for Sp2/0 cells. In theprotoplast fusion method, lysozyme is used to strip cell walls fromcatarrhal harboring the recombinant plasmid containing the chimericantibody gene. The resulting spheroplasts are fused with myeloma cellswith polyethylene glycol.

The chimeric immunoglobulin genes of the present invention can also beexpressed in nonlymphoid mammalian cells or in other eukaryotic cells,such as yeast, or in prokaryotic cells, in particular bacteria.

Yeast provides substantial advantages for the production ofimmunoglobulin H and L chains. Yeasts carry out post-translationalpeptide modifications including glycosylation. A number of recombinantDNA strategies now exist which utilize strong promoter sequences andhigh copy number plasmids which can be used for production of thedesired proteins in yeast. Yeast recognizes leader sequences of clonedmammalian gene products and secretes peptides bearing leader sequences(i.e., pre-peptides) (Hitzman, et al., 11th International Conference onYeast, Genetics and Molecular Biology, Montpelier, France, Sep. 13-17,1982).

Yeast gene expression systems can be routinely evaluated for the levelsof production, secretion and the stability of chimeric H and L chainproteins and assembled chimeric antibodies. Any of a series of yeastgene expression systems incorporating promoter and termination elementsfrom the actively expressed genes coding for glycolytic enzymes producedin large quantities when yeasts are grown in media rich in glucose canbe utilized. Known glycolytic genes can also provide very efficienttranscription control signals. For example, the promotor and terminatorsignals of the phosphoglycerate kinase (PGK) gene can be utilized. Anumber of approaches may be taken for evaluating optimal expressionplasmids for the expression of cloned immunoglobulin cDNAs in yeast (seeGlover, D. M., ed., DNA Cloning, Vol. II, pp. 45-66, IRL Press, 1985).

Bacterial strains may also be utilized as hosts for the production ofantibody molecules or antibody fragments described by this invention. E.coli K12 strains such as E. coli W3110 (ATCC 27325), and otherenterobacteria such as Salmonella typhimurium or Serratia marcescens,and various Pseudomonas species may be used.

Plasmid vectors containing replicon and control sequences which arederived from species compatible with a host cell are used in connectionwith these bacterial hosts. The vector carries a replication site, aswell as specific genes which are capable of providing phenotypicselection in transformed cells. A number of approaches may be taken forevaluating the expression plasmids for the production of chimericantibodies or antibody chains encoded by the cloned immunoglobulin cDNAsin bacteria (see Glover, D. M., ed., DNA Cloning, Vol. I, IRL Press,1985).

Other preferred hosts are mammalian cells, grown in vitro or in vivo.Mammalian cells provide post-translational modifications toimmunoglobulin protein molecules including leader peptide removal,folding and assembly of H and L chains, glycosylation of the antibodymolecules, and secretion of functional antibody protein.

Mammalian cells which may be useful as hosts for the production ofantibody proteins, in addition to the cells of lymphoid origin describedabove, include cells of fibroblast origin, such as Vero (ATCC CRL 81) orCHO-K1 (ATCC CRL 61).

Many vector systems are available for the expression of cloned H and Lchain genes in mammalian cells (see Glover, D. M., ed., DNA Cloning,Vol. II, pp. 143-238, IRL Press, 1985). Different approaches can befollowed to obtain complete H₂L₂antibodies. As discussed above, it ispossible to co-express H and L chains in the same cells to achieveintracellular association and linkage of H and L chains into completetetrameric H₂L₂ antibodies. The co-expression can occur by using eitherthe same or different plasmids in the same host. Genes for both H and Lchains can be placed into the same plasmid, which is then transfectedinto cells, thereby selecting directly for cells that express bothchains. Alternatively, cells may be transfected first with a plasmidencoding one chain, for example the L chain, followed by transfection ofthe resulting cell line with an H chain plasmid containing a secondselectable marker. Cell lines producing H₂L₂ molecules via either routecould be transfected with plasmids encoding additional copies of H, L,or H plus L chains in conjunction with additional selectable markers togenerate cell lines with enhanced properties, such as higher productionof assembled H₂L₂ antibody molecules or enchanced stability of thetransfected cell lines.

In addition to mAbs or chimeric antibodies, the present invention isalso directed to an anti-idiotypic (anti-Id) antibody specific for Vregion epitopes of the mAb antibody or chimeric antibody of theinvention. An anti-Id antibody is an antibody which recognizes uniquedeterminants generally associated with the antigen-binding region ofanother antibody. The antibody specific for CCAA, such as 33.28, istermed the idiotypic or Id antibody. The anti-Id can be prepared byimmunizing an animal of the same species and genetic type mouse strain)as the source of the Id antibody with the Id antibody or theantigen-binding region thereof. The immunized animal will recognize andrespond to the idiotypic determinants of the immunizing antibody andproduce an anti-Id antibody. The anti-Id antibody may also be used as an“immunogen” to induce an immune response in yet another animal,producing a so-called anti-anti-Id antibody. The anti-anti-Id may beepitopically Identical to the original antibody which induced theanti-Id. Thus, by using antibodies to the idiotypic determinants of amAb, it is possible to identify other clones expressing antibodies ofidentical specificity.

Accordingly, the mAbs or chimeric antibodies of the present inventionmay be used to induce anti-Id antibodies in suitable animals, such asBALB/c mice. Spleen cells from such immunized mice can be used toproduce anti-Id hybridomas secreting anti-Id mAbs. Further, the anti-IdmAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH)and used to immunize additional BALB/c mice. Sera from these mice willcontain anti-anti-Id antibodies that have the binding properties of theoriginal mAb specific for a CCAA epitope.

The antibodies of the present invention, including their antigen-bindingfragments and derivatives, have a multitude of uses relating to thediagnosis, monitoring and therapy of colon, breast, and ovarian cancer.Such uses are summarized in Schlom, J., Canc. Res., 46:3225-3238 (1986),which is hereby incorporated by reference.

In diagnosis, the antibodies may be used in immunoassays (describedbelow) to screen body fluids, such as serum, sputum, effusions, urine,cerebrospinal fluid, and the like, for the presence of CCAA. Theantibodies may be used for scanning or radioimaging, when labelled withan appropriate radiolabel, to detect primary or metastatic foci of tumorcells. Furthermore, the antibodies are useful in lymphoscintigraphy todetect lymph node involvement in the disease.

The antibodies of the present invention are also useful forimmunopathological analysis, such as the differential diagnosis of tumortype, the subclassification of the tumor based on its expression ofCCAA. Such determinations would be important in assessment of metastaticpotential, predicted responses to therapy and prognosis.

In particular, because of the specificity of the mAbs and chimericantibodies of the present invention, they may permit the definition ofdefining subpopulations of tumor cells among the heterogeneous cellspresent in a growing tumor. These antibodies could therefore be used inthe typing and cross-matching of the tumor cell “lines” comprising thetumor by means of flow cytometry, both at the time of surgery and priorto therapy. An analysis of the tumor cell subpopulations with theantibodies of this invention, and a battery of additional mAbs, is usedto define (a) which antigen preparation would be the most appropriatefor specific active immunotherapy, (b) which mAb or chimeric antibodywould be efficacious for ADCC, and (c) which antibody or combination ofmAbs should be used for imaging the patient at a later date in searchfor recurrent or metastatic tumors.

In addition to their diagnostic utility, the antibodies of the presentinvention are useful for monitoring the progression of disease byscreening body fluids for CCAA, radioimaging of tumor, or the detectionof occult metastasis through aspiration cytology, lymph node or bonemarrow biopsy, or cytology of body fluids.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes direct cytotoxicity by theantibody, either mediated by complement (CDC) or by effector cells(ADCC), conjugated to anti-tumor drugs, toxins, radionuclides. Theantibodies can be used for ex vivo removal of tumor cells from thecirculation or from bone marrow.

Some of these approaches are described in more detail below. Armed withthe teachings provided herein, one of ordinary skill in the art willknow how to use the antibodies of the present invention for diagnostic,monitoring or therapeutic purposes without undue experimentation.

The preferred animal subject of the present invention is a mammal. Bythe term “mammal” is meant an individual belonging to the classMammalia. The invention is particularly useful in the treatment of humansubjects.

By the term “treating” is intended the administering to subjects of theantibodies of the present invention or a fragment or derivative thereoffor purposes which may include prevention, amelioration, or cure ofcolon, breast, and ovarian cancer.

The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended purpose. Amountsand regimens for the administration of antibodies, their fragments orderivatives can be determined readily by those with ordinary skill inthe clinical art of treating colon, breast, and ovarian cancer andrelated disease.

For example, administration may be by parenteral, subcutaneous,intravenous, intramuscular, intraperitoneal, transdermal, or buccalroutes. Alternatively, or concurrently, administration may be by theoral route. The dosage administered will be dependent upon the age,health, and weight of the recipient, kind of concurrent treatment, ifany, frequency of treatment, and the nature of the effect desired.

Compositions within the scope of this invention include all compositionswherein the antibody, fragment or derivative is contained in an amounteffective to achieve its intended purpose. While individual needs vary,determination of optimal ranges of effective amounts of each componentis within the skill of the art. The effective dose is a function of theindividual chimeric or monoclonal antibody, the presence and nature of aconjugated therapeutic agent (see below), the patient and his clinicalstatus, and can vary from about 10 ng/kg body weight to about 100 10mg/kg body weight. The preferred dosages comprise 0.1 to 10 mg/kg bodyweight.

In addition to the pharmacologically active compounds, the newpharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Preferably, the preparations, contain fromabout 0.01 to 99 percent, preferably from about 20 to 75 percent ofactive compound(s), together with the excipient.

Preparations of the antibody, fragment or derivative of the presentinvention for parenteral administration, such as in detectably labelledform for imaging or in a free or conjugated form for therapy, includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propyleneglycol,polyethyleneglycol, vegetable oil such as olive oil, and injectableorganic esters such as ethyloleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media, parenteral vehicles including sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, such as those based on Ringer's dextrose, and the like.Preservatives and other additives may also be present, such as, forexample, antimicrobials, antioxidants, chelating agents, and inert gasesand the like. See, generally, Remington's Pharmaceutical Science, 16thed., Mack Publishing Co., Easton, Pa., 1980.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating a subject having or developing colon,breast, and ovarian adenocarcinoma. Such treatment comprisesparenterally administering a single or multiple doses of the antibody,fragment or derivative, or a conjugate thereof.

The antibodies of this invention can be adapted for therapeutic efficacyby virtue of their ability to mediate ADCC and/or CDC against cellshaving CCAA associated with their surface. For these activities, eitheran endogenous source or an exogenous source of effector cells (for ADCC)or complement components (for CDC) can be utilized.

The antibodies of this invention, their fragments, and derivatives canbe used therapeutically as immunoconjugates (see for review: Dillman, R.O., Ann. Int. Med. 111:592-603 (1989)). They can be coupled to cytotoxicproteins, including, but not limited to, Ricin-A, Pseudomonas toxin.Diphtheria toxin, and tumor necrosis factor. Toxins conjugated toantibodies or other ligands are known in the art (see, for example,Olsnes, S. et al., Immunol. Today 10:291-295 (1989)). Plant andbacterial toxins typically kill cells by disrupting the proteinsynthetic machinery.

The antibodies of this invention can be conjugated to additional typesof therapeutic moieties including, but not limited to, diagnosticradionuclides and cytotoxic agents such as cytotoxic radionuclides, drugand proteins. Examples of radionuclides which can be coupled toantibodies and delivered in vivo to sites of antigen include 212_(Bi),131_(I), 186_(Re), and 90_(Y), which list is not intended to beexhaustive. The radionuclides exert their cytotoxic effect by locallyirradiating the cells, leading to various intracellular lesions, as isknown in the art of radiotherapy.

Cytotoxic drugs which can be conjugated to antibodies and subsequentlyused for in vivo therapy include, but are not limited to, daunorubicin,doxorubicin, methotrexate, and Mitomycin C. Cytotoxic drugs interfacewith critical cellular processes including DNA, RNA, and proteinsynthesis. For a fuller exposition of these classes of drugs which areknown in the art, and their mechanisms of action, see Goodman, A. G., etal., Goodman and Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7thEd., Macmillan Publishing Co., 1985.

The antibodies of this invention may be advantageously utilized incoordination with other monoclonal or chimeric antibodies, or withlymphokines or hemopoietic growth factors, etc., which serve to increasetee number or activity of effector cells which interact with theantibodies.

The antibodies, fragments, or derivatives of this invention, attached toa solid support, can be used to remove soluble coloncarcinoma-associated antigens from fluids or tissue or cell extracts. Ina preferred embodiment, they are used to remove soluble tumor antigensfrom blood or blood plasma products. In another preferred embodiment,the antibodies are advantageously used in extracorporeal immunoadsorbentdevices, which are known in the art (see, for example, Seminars inHematology, Vol. 26 (2 Suppl. 1) (1989)). Patient blood or other bodyfluid is exposed to the attached antibody, resulting in partial orcomplete removal of circulating CCAA (free or in immune complexes), ofCCAA-bearing cells, following which the fluid is returned to the body.This immunoadsorption can be implemented in a continuous flowarrangement, with or without interposing a cell centrifugation step.See, for example, Terman, D. S. et al., J. Immunol. 117:1971-1975(1976).

The present invention also provides the above antibodies, fragments andderivatives, detectably labelled, as described below.

The antibodies of the present invention are useful for immunoassayswhich detect or quantitate CCAA or cells bearing CCAA in a sample. Suchan immunoassay typically comprises incubating a biological sample in thepresence of a detectably labelled antibody of the present inventioncapable of identifying the tumor antigen, and detecting the labelledantibody which is bound in a sample.

Thus, in this aspect of the invention, a biological sample may betreated with nitrocellulose, or other solid support or carrier which iscapable of immobilizing cells, cell particles or soluble proteins orglycoproteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labelled antibody of thepresent invention. The solid phase support may then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on said solid support may then be detected by conventional means.

By “solid phase support” or “carrier” is intended any support capable ofbinding antigen or antibodies. Well-known supports or carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses,and magnetic. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toCCAA or the antibody specific for CCAA. Thus, the support configurationmay be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of antibody may be determinedaccording to well-known methods. Those skilled in the art will be ableto determine operative and optimal assay conditions for eachdetermination by employing routine experimentation.

One of the ways in which the antibody of the present invention can bedetectably labelled is by linking the same to an enzyme and use in anenzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA).This enzyme, when subsequently exposed to its substrate, will react withthe substrate generating a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or by visual means. In analternate embodiment, the enzyme is used to label a binding partner forthe antibody of the invention. Such a binding partner may be an antibodyagainst the constant or variable region of the antibody of theinvention, such as a heterologous anti-mouse immunoglobulin antibody.Alternatively, the binding partner may be a non-antibody protein capableof binding to the antibody of the present invention, such asstaphylococcal protein A, or streptococcal protein G.

Enzymes which can be used to detectably label the CCAA-specificantibodies of the present invention, or the binding partners for theseantibodies, include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glycoamylase andacetylcholinesterase.

By radioactively labelling the antibody of the present invention or thebinding partner, it is possible to detect CCAA through the use of aradioimmunoassay (RIA) (see, for example, Work, T. S. et al., LaboratoryTechniques and Biochemistry in Molecular Biolog. North HollandPublishing Company, N.Y. (1978)). The radioactive isotope can bedetected by such means as the use of a gamma counter or a scintillationcounter or by autoradiography. Isotopes which are particularly usefulfor the purpose of the present invention are well known in the art.

It is also possible to label the antibodies or binding partners with afluorescent compound. When the fluorescently labelled antibody isexposed to light of the proper wave length, its presence can then bedetected due to fluorescence. Among the most commonly used fluorescentlabelling compounds are fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andflourescamine.

The antibodies can also be detectably labelled usingfluorescence-emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the antibody using such metalchelating groups as diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

The antibodies of the present invention also can be detectably labelledby coupling to a chemiluminescent compound. The presence of thechemiluminescently labelled antibody is then determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Examples of particularly useful chemiluminescent labellingcompounds are luminol, isoluminol, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody,fragment or derivative of the present invention. Bioluminescence is atype of chemiluminescence found in biological systems, in which acatalytic protein increases the efficiency of the chemiluminescentreaction. The presence of a bioluminescent protein is determined bydetecting the presence of luminescence. Important bioluminescentcompounds for purposes of labelling are luciferin, luciferase andsequorin.

Detection of the antibody, fragment or derivative may be accomplished bya scintillation counter, for example, if the detectable label is aradioactive gamma emitter, or by a fluorometer, for example, if thelabel is a fluorescent material. In the case of an enzyme label, thedetection can be accomplished by colorimetric methods which employ asubstrate for the enzyme. Detection may also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate incomparison with similarly prepared standards.

In situ detection may be accomplished by removing a histologicalspecimen from a patient, and providing the labelled antibody, or theunlabelled antibody plus a labelled binding partner to such a specimen.Through the use of such a procedure, it is possible to determine notonly the presence of the antigen but also its distribution in theexamined tissue. Using the present invention, those of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection. Such methods include, for example,immunohistochemical staining procedures. In a preferred embodiment, anavidin-biotin immunoperoxidase staining system can be used, and a kitutilizing this system is also contemplated.

The kit employing mAbs or chimeric antibodies of the present inventioncan be used for immunohistochemical evaluation of colon, breast, andovarian carcinoma. Indications for tissue study are to evaluatesubpopulations of tumor cells that express the antigens defined by mAbs31.1 and 33.28.

The colon kit is comprised of the reagents necessary forimmunohistochemical analysis as follows:

-   -   a) mAbs 31.1, 33.28 or mouse/human chimeric antibody Chi #1, and        the mAb for carcinoembryonic antigen (CEA), the latter        representing the standard monoclonal used for colon tissue        immunohistochemistry;    -   b) reagents for immunoperoxidase (blocking reagent) in the form        of, for example, goat serum; and secondary antibody, such as,        for example, goat anti-mouse antibody;    -   c) immunoperoxidase; and    -   d) reagents to produce the brown coloration.

Similar kits can be employed for the immunohistochemical analysis ofbreast and ovarian carcinoma.

The immunoperoxidase technique to be employed is that of Sternberger.The primary antibody (mAb or chimeric antibody) serves as an antigenwhich can bind more than one secondary antibody. The secondaryantibodies form a “bridge” between the primary antibody and thehorseradish peroxidase-antiperoxidase complexes.

The kit contemplated herein can be used to study fully developed colon,carcinoma, polyps in transformation to define the extent of malignanttransformation, benign polyps to see if a site of transformation hasbeen missed and inflammatory bowel disease to evaluate any sites ofundetected transformation. Similar kits can be employed to study breastand ovarian carcinomas.

Another kit similar to the above kit is also contemplated which uses allfive mAbs to colon carcinoma in order to evaluate all subpopulations oftumors and as such has the capability to type and cross-match thelesions.

The antibody, fragment or derivative of the present invention may beadapted for utilization in an immunometric assay, also known as a“two-site” or “sandwich” assay. In a typical immunometric assay, aquantity of unlabelled antibody (or fragment of antibody), is bound to asolid support that is insoluble in the fluid being tested and a quantityof detectably labelled soluble antibody is added to permit detectionand/or quantitation of the ternary complex formed between solid-phaseantibody, antigen, and labelled antibody.

Typical, and preferred, immunometric assays include “forward” assays inwhich the antibody bound to the solid phase is first contacted with thesample being tested to extract the tumor antigen from the sample byformation of a binary solid phase antibody-CCAA complex. After asuitable incubation period, the solid support is washed to remove theresidue of the fluid sample, including unreacted tumor antigen, if any,and then contacted with the solution containing an unknown quantity oflabelled antibody (which functions as a “reporter molecule”). After asecond incubation period to permit the labelled antibody to complex withthe CCAA bound to the solid support through the unlabelled antibody, thesolid support is washed a second time to remove the unreacted labelledantibody. This type of forward sandwich assay may be a simple “yes/no”assay to determine whether CCAA is present or may be made quantitativeby comparing the measure of labelled antibody with that obtained for astandard sample containing known quantities of the antigen. Such“two-site” or “sandwich” assays are described by Wide (Radioimmune AssayMethod, Kirkham, ed., E. & S. Livingstone, Edinburgh, 1970, pp.199-206).

Other type of “sandwich” assays, which may also be useful with CCAA, arethe so-called “simultaneous” and “reverse” assays. A simultaneous assayinvolves a single incubation step wherein the antibody bound to thesolid support and labelled antibody are both added to the sample beingtested at the same time. After the incubation is completed, the solidsupport is washed to remove the residue of fluid sample and uncomplexedlabelled antibody. The presence of labelled antibody associated with thesolid support is then determined as it would be in a conventional“forward” sandwich assay.

In the “reverse” assay, stepwise addition first of a solution oflabelled antibody to the fluid sample, followed by the addition ofunlabelled antibody bound to a solid support after a suitable incubationperiod, is utilized. After a second incubation, the solid phase iswashed in conventional fashion to free it of the residue of the samplebeing tested and the solution of unreacted labelled antibody. Thedetermination of labelled antibody associated with a solid support isthen determined as in the “simultaneous” and “forward” assays. In oneembodiment, a combination of antibodies of the present inventionspecific for separate epitopes may be used to construct a sensitivethree-site immunoradiometric assay.

For purposes of in vivo imaging of colon, breast, and ovarian cancerusing the antibodies of the present invention, there are many differentlabels and methods of labelling known to those of ordinary skill in theart. Examples of the types of labels which can be used in the presentinvention include radioactive isotopes, paramagnetic isotopes, andcompounds which can be imaged by positron emission tomography (PET).Those of ordinary skill in the art will know of other suitable labelsfor binding to the antibodies used in the invention, or will be able toascertain such, using routine experiments. Furthermore, the binding ofthese labels to the antibody can be done using standard techniquescommon to those of ordinary skill in the art.

For diagnostic in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given radionuclide. Theradionuclide chosen must have a type of decay which is detectable by agiven type of instrument. In general, any conventional method forvisualizing diagnostic imaging can be utilized in accordance with thisinvention.

Another important factor in selecting a diagnostic radionuclide for invivo imaging is that the half-life of a radionuclide be long enough sothat it is still detectable at the time of maximum uptake by the targetissue, but short enough so that deleterious radiation of the host isminimized. In one preferred embodiment, a radionuclide used for in vivoimaging does not emit particles, but produces a large number of photonsin a 140-200 keV range, which may be readily detected by conventionalgamma cameras.

For in vivo diagnosis, radionuclides may be bound to the antibody eitherdirectly or indirectly by using an intermediary functional group.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to the antibodies are thechelating agents, diethylene triamine pentaacetic acid (DTFA) andethylene diamine tetraacetic acid (EDTA). Examples of metallic ionswhich can be bound to the antibodies of the present invention are^(99m)Tc, ¹²³I, ¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, ¹²⁵I, ⁶⁸Ga, ⁷²As, ⁸⁹Zr,and ²⁰¹TI.

The specifically exemplified mAbs 33.28 and 31.1, and the chimericantibody Chi #1 may be used to facilitate the production of additionalmAbs which bind the same or immunologically cross-reactive coloncarcinoma-associated antigens. First, these antibodies may be conjugatedto a chromatographic support, and used to immunopurify coloncarcinoma-associated antigens. These purified antigens, in turn, may beused to stimulate an immune response in suitable animals. Secondly,spleen cells from the responsive animals may be fused to immortalizingcells, and the resulting hybridomas screened for secretion of antibodieswhich bind to the purified antigen and/or whose binding to coloncarcinoma-associated antigen is competitively inhibited by antibody33.28 or 31.1, or chimeric antibody Chi #1.

Having now generally described the invention, the same will be furtherunderstood by reference to certain specific examples which are includedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLE I Preparation and Characterization of the ColonCarcinoma-Associated Antigen (CCAA)

The antigenic preparation was obtained from pooled colon carcinomamembranes according to the method described by Hollinshead et al.,Cancer 56:480 (1985). This antigenic material was purified to the extendthat the membrane fractions were free of HL-A antigens and wereseparated from much of the non-immunogenic glycoprotein fractions. Inits final form the antigenic preparation was shown to be immunogenic ina specific manner in humans as evidenced by its capability of elicitinga delayed hypersensitivity reaction only in patients with active colon,breast, and ovary carcinoma.

Tumor cell suspensions in saline were prepared from fresh operating roomspecimens. Single cell suspensions, obtained by conventional means, werecentrifuged for 10 minutes at about 400×g and the supernatant wasretained. The cell pellet was resuspended and recentrifuged. Themembrane material was examined by electron microscopy to assure thatonly membrane material (and no intact cells) was present, and theprotein content was measured by the Lowry method.

The membrane material was next subjected to sequential low frequencysonication and resuspended as a soluble pool of membrane proteins. Thesoluble sonicates were separated by gel filtration on Sephadex-6200.Fractions of 2 ml were collected and the absorbance profile at 220 and280 μm was recorded. Fractions comprising individual protein peaks werepooled, and the pools were concentrated by Diaflo ultrafiltration.Sephadex-G200 fractions IB and IIA, as defined by Hollinshead et al.,(supra), were further purified by gradient polyacrylamide gelelectrophoresis (PAGE). The fractions were tested for their ability toelicit positive delayed cutaneous hypersensitivity reactions in patientswith colon carcinoma. Those fractions with immunogenic activity weresaid to contain colon carcinoma-associated antigens (CCAA) and wereemployed as immunogens and screening agents in the preparation of themAbs.

By gradient PAGE, a double-banded antigen distinct from that ofcarcinoembryonic antigen (Gold, P. et al., J. Exp. Med. 122:467-481(1965); Hollinshead, A. et al., Cancer 56:480 (1985)) was identified andisolated. The bands comprising this antigen migrated 6.3 and 6.6 cm.distant from tracking dye. Biochemical analysis of the antigen provedthat it was glycoprotein. The molecular weight of the antigen wasestimated based on the electrophoretic mobility of transferrin (6.4-6.5cm) which has a molecular weight of 76.5 kDa.

EXAMPLE II Preparation and Screening of Monoclonal Antibodies

Monoclonal antibodies (mAbs) against human colon carcinoma-associatedantigens (CCAA) were obtained by the production and cloning of hybridsresulting from the fusion of mouse myeloma cells Sp2/0-Ag14 with spleencells from BALB/c mice which had been immunized with the CCAA describedabove.

Five hybrid clones were established, as described below, and designatedas 31.2, 31.1, 77, 33.23 and 33.28. All five mAbs reacted strongly withthe CCAA and with two colon carcinoma cell lines (SW480 and SW620) whenassayed by ELISA. Two of the mAbs, 31.1. and 33.28, were studied ingreatest detail.

A. Immunization and Cell Fusion

BALB/c mice were immunized by intrapertioneal injection of 50 μg of theCCAA described above emulsified in complete Freud's adjuvant, asdescribed by Hollinshead in clinical trials (Hollinshead et al., supra).Ten days later the mice received an intravenous booster injection of thesame amount of CCAA in saline. Mice were sacrificed three days later andtheir spleen cells obtained. Cell fusion was performed by incubation5×10⁷ mouse spleen cells with 10⁷ sP2/0-Ag14 myeloma cells in 40%polyethylene glycol (MW=1500)

B. Screening of Hybrid Clones

An enzyme-linked immunosorbent assay (ELISA), described by Tsang et al.,JNCI77:1175 (1986), was used for the detection of hybridoma clonesproducing antibodies specific for the CCAA. CCAA (100 n/well) wasimmobilized on polystyrene microplates. Antibodies present in the testsupernatants were allowed to bind to the immobilized antigen. Thepresence of the bound murine mAbs was detected withperoxidase-conjugated second antibodies, specific for mouseimmunoglobulins, followed by the chromogenic substrate for peroxidase,O-phenyldiamine. Wells showing color reactions yielding Absorbances≧0.500 units were scored as positive. Negative controls gave values of0.01 to 0.09 units.

Hybridoma wells scoring as positive by ELISA were further screened byindirect immunofluorescene, using various tumor cells and normal cellsas identified in Table 1, below. All of the tumor cell lines wereobtained from the ATCC. Cells were incubated with hybridoma culturesupernatants at an appropriate dilution (1:2) in phosphate bufferedsaline (PBS) for 1 hour at 4° C. The cells were washed and incubatedwith a fluorescein-labelled goat anti-mouse immunoglobulin antibody. Thecells were then washed three times with PBS and examined by fluorescencemicroscopy. The results appear in Table 1.

TABLE 1 INDIRECT IMMUNOFLUORESCENCE REACTIVITY OF ANTI-COLONCARCINOMA-ASSOCIATED ANTIGEN(S) (COAA) MoAbS WITH HUMAN CULTURECELLS^(A) REACTIVITY OF MoAbS^(B) CELLS 31.2 77 31.1 33.28 33.23 TUMORLINES SW948 (COL) − + − − − HCT116 (COL) − − − + − WIDR (COL) + + + + +COLO320 (COL) + + + − − HS619 (COL) − − − − − HS853 (COL) − − − − −CACO-2 (COL) + + − − − SK-CO-1 (COL) + + − + + HT-29 (COL) + + − + +SW1116 (COL) + − − + + SW480 (COL) + + + + + SW620 (COL) + + + + + 231(BE) − − − − − CAMA-1 (BE) − − − − − PAN-1 (PAN) − − + − − MIA (PAN) −− + − − HS766T (PAN) − − − − − M-14 (MEL) − − − − − HT1080 (FIB) − − − −− LM (OS) − − − − − TE-85 (OS) − − − − − NORMAL SKIN − − − − −FIBROBLAST BONE MARROW − − − − − CELL NORMAL HUMAN − − − − − PBMC^(A)CULTURE SUPERNATANT WAS DILUTED 1:2 WITH PBS. ^(B)POSITIVE (+) ANDNEGATIVE (−) REACTIVITIES WERE DEFINED BY INTENSITY OF THE MEMBRANEFLUORESCENCE AS COMPARED TO THE BACKGROUND. C. COL: COLON CARCINOMA; BR:BREAST CARCINOMA; PAN: PANCREATIC CARCINOMA; FIB: FIBROSOMCOMA.

EXAMPLE III Analysis of Monoclonal Antibodies and their Reactivity

The anti-CCAA mAbs produced and detected as above were also tested forreactivity with fresh human tissue. Cryostat sections of the tissuetypes listed in Table 2, below, were fixed with 3.5% formaldehyde in PBSand then washed three times with PBS. For indirect immunofluorescencestudies, the sections were incubated with the mAbs and then stained witha fluorescein-labelled second antibody as above.

As is shown in Table 2, mAbs 31.1 and 33.28 were highly specific forcolon carcinoma cells. This indicates that an antigen (CCAA) which washighly specific for colon carcinoma and, furthermore, was immunogenic incolon carcinoma patients (positive DH reactions), and served as asuccessful immunogen in mice for the devnd phycoerythrin excitation wasused. Trigger regions were established by examining cells by forwardversus 90° light scatter. As shown in Table 4, both 31.1 and 33.28 boundto colon carcinoma cells; neither mAb bound significantly to PBMC.

TABLE 2 Indirect Immunofluorescence of Anti-CCAA Mabs with Flesh HumanTissues^(a) Reactivity of Mabs Tissues 33.28 31.1 Tumor Colon Carcinoma3/3 3/3 Pancreatic Carcinoma 0/2 0/2 Melanoma 0/2 0/1 Breast Carcinoma0/2 0/1 Normal Placenta 0/1 0/1 Liver 0/1 0/1 Colon 0/3 0/3 Spleen 0/10/1 Thymus 0/1 0/1 Muscle 0/1 0/1 ^(a)Ascitic fluid was diluted 1:50with PBS. Cryostat sections (4-6 μM thick) were fixed with 3.5%formaldehyde in PBS for 10 minutes and then washed three times in PBS.Sections were stored at −70° C. unless used immediately. Results areexpressed as number of positive/negative of tissue tested.

Table 3 shows the results of an immunoabsorption analysis of the Mabs.Three colon carcinoma cell lines (HT-29, WIDR and SW 620) and anosteosarcoma cell line (LM) were used to absorb fluoresceinisothiocyanate (FITC)-conjugated Mabs 31.1 or 3.28. Ascites fluid frommice in which the hybridomas were growing, diluted 1:50, was added tothe absorbing cells. The mixtures were incubated for 1 hr at 4° C.Either 2×10⁷ cells (Table 3, Part A) or 10⁴ cells (Table 3, Part 5) wereused to absorb the antibodies (Table 3) The osteosarcoma cell line didnot absorb out 31.1 or 33.28 activity, while the colon carcinoma celllines did.

TABLE 3 Immunoabsorption Analysis of mAbs Absorbing Cells HT-29 WIDRSW620 LM mAb A B A B A B A B 33.28 − + − + − + + + 31.1 − + − + − + + +

Cytofluorometric analysis was used to measure the binding of 31.1. 33.28and a control mAb to HT29, WIDR and SW480 tumor cells and to peripheralblood mononuclear cells (PBMCs) An Ortho Spectrum III Cytofluorograph,equipped with an argon laser capable of fluorescein and phycoerythrinexcitation was used. Trigger regions were established by examining cellsby forward versus 90° light scatter. As shown in Table 4, both 31.1 and33.28 bound to colon carcinoma cells; neither mAb bound significantly toPBMC.

TABLE 4 SUMMARY OF CYTOFLUOROMETRIC ANALYSIS % of cells stained withmAb; Cells 33.28 31.1 Control HT29 51.0 53.9 9.2 WiDr 21.0 ND 8.1 SW48037.0 32.0 3.8 PBMC 2.1  2.1 ND

The heavy and light chain isotypes of the mAbs were determined byimmunodiffusion. The 31.1 mAb was found to be an IgG1 with a kappa lightchain. The 33.28 mAb was found to be an IgG2a with a kappa light chain(Table 5). This is in strong contrast to the prior art mAb 19.9 (Herlyn,M. et al., J. Biol. Chem. 257:14365-14369 (1982)) which is of the IgG1class (Herlyn, D. et al., Proc. Natl. Acad. Sci. USA 79:4761-4765(1982)). Importantly, antibodies of the IgG2a class are expected to bemore useful for immunotherapeutic purposes (Colcher, D. et al., Proc.Natl. Acad. Sci. USA 78:3199-3203 (1981)). Although the 19.9 mAb hasreactivity to colon tumors, it was derived by immunization withpancreatic carcinoma antigens, and is therefore cross-reactive withcolon. This is analogous to the situation with the B72.3 mAb([citation]), which is a colon-reactive antibody obtained byimmunization with breast cancer tissue.

TABLE 5 Isotyping of Monoclonal Antibodies Culture Light ChainsSupernatant IgG1 IgG2a IgG2b IgM Kappa Lambda 31.2 − + − − + − 31.1 + −− − + − 77 − − + − + − 33.23 − + − − + − 33.28 − + − − + −

EXAMPLE IV Characterization of the Colon Carcinoma-Associated Antigen

The molecular mass of the antigens to which the above mAbs bound wasdetermined by Western blot analysis using soluble protein extracted fromcolon carcinoma cell lines SW480 and SW620. The 33.28 and 31.1 mAbsantibody reacted with molecules having an apparent molecular weightsweight of 61.1 kDaand 72 kDa, respectively , from both of these cellslines. These mAbs The 33.28 and 31.1 mAbs did not react with materialfrom human PBMCs or from human tumor cell lines of other histologictypes in Western blot analysis.

In order to define better the specificity of the mAbs of the presentinvention for the immunizing CCAA and to establish whether the mAbsreacted with an immunogenic component of the cell membrane preparationwhich has been used in clinical immunotherapy trials (Hollinshead etal., supra), the original immunogenic preparation described above wasperformed by high performance liquid chromatography (HPLC).

The analysis revealed 4 distinct peaks, each of which was tested forimmune reactivity (elicitation of DH) in patients with colon carcinomaby skin test (FIG. 1). Among the 10 patients with colon carcinoma testedonly the material in peak #4 induced a cutaneous DH reaction. The peak#4 antigen was found to react with mAb 33.28, while mAb 31.1 reactedwith peak #3, the next most prominent peak.

The references cited above are all incorporated by reference herein.

EXAMPLE V Affinity Purification of Colon Carcinoma-Associated Antigen

The mAb 33.28 was used in affinity chromatography to isolate antigenextracted from cells of the HT-29 line. Five mg of purified 33.28 IgGwas coupled to CNBr-activated sepharose 4B. The column was pre-elutedwith 0.05M diethylamine, pH 11.5, and then equilibrated with 0.14MNaCl/0.01M Tris (pH 8.0). CCAA preparation was applied to the column,and the column was eluted with 0.05M diethylamine, pH 11.5. The elutedfractions were neutralized by the addition of IM Tris-HCl, pH 8.0.

The material bound and eluted from the 33.28 affinity matrix was thensubjected to HPLC. The eluted CCAA preparation was adjusted in startingbuffer (0.01M sodium phosphate buffer, pH 7.0), applied to a SynchropakWax weak anion exchange HPLC column (250×4.6 mm) and eluted with agradient of 0 to 1M NaCl in 0.01M sodium phosphate buffer, pH 6.0, at aflow rate of 1 ml/min. Anion exchange chromatography was performed usinga Hewlett-Packard HPLC (HP 1090, Hewlett-Packard, Arondale, Pa.).

Results appear in FIG. 2. The antigenic material derived from HT-29cells isolated by mAb 33.28 gave a peak that matched peak #4 describedabove and had similar immunogenic activity in humans, indicating theutility of mAb 33.28 for isolation of a colon cancer preparation whichis immunogenic for humans.

EXAMPLE VI ADCC Activity of mAbs 33.28 and 31.1

In order to be therapeutically useful, a mAb specific for an immunogenictumor antigen should have the following properties: (a) high tumortissue specificity, (b) absence of cross-reactivity to normal humantissue, and (c) a biological activity associated with destruction oftumors, such as antibody-dependent cellular cytotoxicity (ADCC).

The ADCC activity of mAbs 33.28 and 31.1 was tested on the coloncarcinoma line WiDR as target cell. The melanoma cell line, M-14, servedas a specificity control. ADCC was assayed using a conventional 4 hr.⁵¹Cr release assay using normal human PBMC as effector cells, and theresults are shown as percent isotope release (% lysis) (Table 6). Thebackground lysis was 8.3%. At an effector:target ratio of 100:1, mAb33.28 caused 40.3% lysis of tumor cells, and 31.1 induced 51.8% lysis.

TABLE 6 ADCC Activity of mAbs 33.28 and 31.1 % Lysis of Target Cells atE:T Ratios: Antibody WiDR M-14 or Control 25 50 100 25 50 100 33.28 23.140.3 45.3 6.9 8.4 9.0 31.1 14.3 26.7 51.8 7.5 6.4 8.7 OSA1 10.0 9.2 12.211.4 14.8 10.9 NMS 12.2 11.7 13.1 14.2 15.0 11.1 PBS 8.2 5.1 7.6 11.014.2 10.5 ADCC was assayed by a 4 hour ⁵¹Cr release assay. Background⁵¹Cr release was 8.3%. E:T Ratio indicates effector cell-to-targetratios. The mAb or serum was tested at a 1:100 dilution; OSA1 - mAbto-osteosarcoma associated antigens; NMS - normal mouse serum; WiDr -colon carcinoma cell line; M14 - melanoma cell line.

EXAMPLE VII Detection of Circulating CCAA with mAbs 33.28 and 31.1

The mAbs of the present invention were tested for their ability todetect circulating CCAA in 79 unknown serum samples (Table 7). The assaywas based on the ability of the serum samples to inhibit binding of themAb to the CCAA in ELISA. None of the 50 normal serum samples gave falsepositive results. Nine of the ten serum samples from patients withactive colon carcinoma were positive. None of the sera from disease-freecolon cancer patients one year post-resection were positive.

TABLE 7 Detection of Circulating Colon Carcinoma-Associated Antigen No.of sera inhibiting binding of mAbs; 33.28 31.1 DONOR No. of <15% >15%<15% >15% CONDITION Samples (Neg) (Pos) (Neg) (Pos) Colon 10 3 7 2 8Carcinoma Colon 4 4 0 4 0 Carcinoma (Resected) Breast 9 9 0 9 0Carcinoma Melanoma 5 5 0 5 0 Prostate 1 1 0 1 0 Cancer Normal Serum 5050 0 50 0 Colon carcinoma-associated antigens was detected by ELISA. 100μl of serum were used in each assay.

EXAMPLE VIII Comparison of Specificity with Other mAbs Reactive withColon Tumors

Further studies of tumor specificity were conducted using ELISA (Table8). The mAb 31.1 was compared with CC49, a colorectal carcinoma-specificmAb purified from B-72.3, and a control mouse myeloma protein. 31.1 wasshown to react with a narrower range of colorectal carcinomas than didCC49. However, it had a higher degree of specificity, having lower or nocross-reactivity with stomach tumors or normal colon tissue.

TABLE 8 ELISA ON NORMAL AND TUMOR TISSUES USING MAbs 31.1 CC49 ANDMOPC-21 Tissues 31.1 CC49 MOPC-21 Colorectal Carcinomas  1. COCA2A − +++−  2. COCA2 − +++ −  3. COCA3 + ++ −  4. COCA4 +++ +++ −  5. G820 ± ++ − 6. G853 +++ ++ −  7. G817 +++ +++ −  8. G781 − − − Other Carcinomas  1.Breast CA1 − − −  2. Breast CA2 − − −  3. Lung CA1 − − −  4. Lung CA2 −− −  5. Ovarian CAD106 − − −  6. Ov CA5 − − −  7. Ov CAV5 − − −  8. OvCAV45 − − −  9. Ov CAV43 − − − 10. Stomach CA14A ++ +++ 11. StomachCA12A − +++ − 12. Stomach CA15A − − − Other Normal Tissues  1.Endometrium E21 − − −  2. Endometrium EC19 − − −  3. Endometrium EC17− + −  4. Endometrium EC18 − − −    (RBC)  5. Red blood cells 1 − − − 6. RBC 2 − − −  7. RBC 3 − − −  8. RBC 4 − − −  9. RBC 5 − − − 10. RBC6 − − − 11. RBC 7 − − − 12. RBC 8 − − − 13. RBC 9 − − − 14. RBC 10 − − −15. RBC 11 − − − 16. Granulocytes − − − 17. 385 − − − 18. 386 − − − 19.Normal spleen 3 − − − 20. 392 (N. Spleen) − − − 21. 395 (N. Liver) − − −22. 387 (N. Kidney) − − − 23. 398 (N. Spleen) − − − 24. 390 (N. Liver) −− − 25. N. Spleen #1 − − − 26. N. Spleen #2 − − − 27. 800 (N. Colon) −++ − 28. N. Colon (GW) − ++ − 29. N. Colon (Meloy) − − − 30. N. Colon −− − 31. G1155B (N. Colon) − − − 32. G1164B (N. Colon) − − − 33. N. Colon± − − 34. Normal Stomach A − − − 35. N. Stomach B − − − 36. N. Stomach C− − − 37. Normal Lung − − − 38. Normal Liver − − − CC49 - NCI monoclonalantibody to colorectal carcinomas MOPC-21 - negative control myelomaprotein All monoclonal antibodies were used at 40 ng/well, POGAM at1:3000 dilution

EXAMPLE IX In Vivo Localization of mAbs 33 28 and 31.1 to Tumors

The in vivo behavior of the mAbs of the present invention was examinedby pharmacokinetic studies using ¹²⁵I-labelled mAb and athymic nude micebearing LS-174T colon tumor xenografts. Mice implanted with the A375melanoma were employed as controls. The relative concentration of mAb intumor as compared to adjacent normal tissue such as liver and spleen isrepresented by the radiolocalization index (concentration ofradiolabelled material in tumor/concentration in surrounding tissue).The biodistribution of ¹²⁵I-labelled 31.1 and 33.28 mAbs is shown inTable 9. Both mAbs were able to significantly concentrate within thetumor, compared to localization in normal tissues (spleen and liver).This selective accumulation was six-fold at 96 hours and about 12-foldat 168 hrs. The radiolocalization indices for both mAbs to tumor ascompared to blood, liver and spleen are shown in FIG. 3 and FIG. 4.

TABLE 9 Biodistribution of ¹²⁵I-mAbs in Tumor-Bearing Athymic Nude Mice96 hours 168 hours Tissue LS174T A375 LS174T A375 A. mAb 31.1 Blood 7.30NA 4.67 4.42 Tumor 21.92 NA 25.43 2.91 Liver 3.74 NA 2.16 1.24 Spleen3.68 NA 2.41 1.32 B. mAb 33.28 Blood 7.81 NA 5.58 3.95 Tumor 13.12 NA15.50 2.24 Liver 2.55 NA 1.74 1.68 Spleen 2.31 NA 1.70 1.92 Results areexpressed in % injected dose/gram of tissue. LS174T = colon carcinoma;A375 = melanoma.

EXAMPLE X Immunohistochemical Studies

The ability to detect tumor markers in the serum, image the relatedneoplastic process and define the cell population of that neoplasm byimmunohistochemistry depends on the ability of specific mAbs toselectively characterize a tumor population.

The mAbs 31.1 and 33.28 were tested in more than 50 colon carcinomas bymeans of immunoperoxidase staining. They have been found to be highlyreactive with the colon neoplasm and did not interact with the adjacentnormal tissues. When polyps were evaluated, the wholly benign lesionssuch as villo-tubular adenomas showed no reactivity. Villous adenomasundergoing transformation reacted only at the site of malignancy. Whensimilar tissues were evaluated using the more commoncarbohydrate-antigen derived mAbs, normal adjacent colon tissue reactedequally with the neoplastic portion of the specimen. With the 31.1 and33.28 mAbs, each appeared to stain different cell populations within thetumor. This suggests that surface antigens representing differentoncogene products were being defined.

EXAMPLE XI Selective Binding to Subpopulations of Epithelial Cells inParaffin-Embedded Benign and Malignant Mammary Lesions

Forty-one formalin-fixed, paraffin-embedded benign and malignant breastspecimens were studied with mAbs 31.1 and 33.28, using the avidin-biotinstaining method. Without enzymatic pretreatment, positive staining ofepithelium was observed on the cell surface and in the cytoplasm withboth antibodies. 7/21 (33%) duct carcinogens were positive with mAb 31.1as were 5/20 (25%) samples of benign breast disease. 10/21 (48%) ductcarcinomas were positive with mAb 33.28 together with 7/20 (35%)specimens of benign mammary disease. 10 to 75% of the cell populationwas positively stained. These results indicate that the antigens definedby mAbs 31.1 and 33.28 are expressed in a select group of women withbreast disease and would be useful for diagnosis of said disease.

EXAMPLE XII Selective Binding to Subpopulations of Epithelial Cells inFresh Frozen Benign and Malignant Ovarian Tumors

Fresh frozen tissue biopsies obtained from twenty-one ovarian tumorssubjected to immunocytochemical analysis were studied with mAbs 31.1 and33.28 using the avidin biotin indirect immunoperoxidase assay. Focalpositive staining was observed in 4/7 papillary mucous, 1/1 mucinous and½ endometroid adenocarcinoma. None of the nonepithelial ovarian tumorsstained positive using these monoclonal antibodies. These resultsindicate that the antigens as defined by mAbs 31.1 and 33.28 areexpressed in a select group of woman with ovarian cancer and would beuseful for diagnosis of said disease.

EXAMPLE XIII Immunoreactivity of Human Carcinoma Tissues and Cell Lineswith mAbs 31.1 and 33.28

The mAbs 31.1 and 33.28 were used to screen a panel of cell linesincluding colon adenocarcinoma, lymphoma, leukemia and neuroblastomalines.

Using the avidin-biotin immunoperoxidase staining system, the mAbs 31.1and 33.28 were shown to strongly bind to colon adenocarcinoma cell linesWIDR and HT-29. Immunoreactions were not observed with KG1-a, HL-60,Molt-3 and JUKRAT cell lines. Both antibodies reacted weakly with onelymphoma line (JY). The mAb 33.28 reacted weakly with one leukemic line(K562) and a neuroblastoma line (U87. MG). These results confirm andextend previous flow cytometry and immunofluorescent results in which ithas been reported that strong binding reactions were observed with thesemAbs with colon adenocarcinoma cell lines and reactions were notobserved with other tumor cell lines. Using flow cytometry, mAb 31.1reacted with 85% of HT-29 and WIDR colon carcinoma cells but not withSKBR-3 breast cancer cells.

Both antibodies were extensively shown to bind distinctively to coloncarcinoma tissues (mAb 33.28-84%, mAb 31.1-64%), and not to normaltissues or malignant tissues including neuroblastoma tissues ( 0/3),lymphomas ( 0/3) and leukemic infiltrates ( 0/3) tested. These resultssuggest that these antibodies can serve as a useful research tool inevaluating tumor markers in cancer and cell biology research.

EXAMPLE XIV Expression and Characterization of Chimeric AntibodiesAgainst Human Colorectal Carcinoma-Associated Antigen

A chimeric mouse/human heavy chain gene was constructed by splicing theexon of the 31.1 antibody heavy chain variable region gene to the exonof the human gamma1 chain contstant region gene using the polymerasechain reaction. Subsequently, the 31.1 chimeric gene was cloned into aretroviral expression vector pLgptCXII and transfected into thepackaging cell line PA317. The transfected cells (PA317H) werecultivated with another packaging cell line PA317L, which contained anirrelevant mouse/human chimeric light chain gene in retroviralexpression vector pLneoCXII, and SP2/0-Ag14 cells. The transducedSP2/0-Ag14 cells yielded a complete chimeric antibody. Chi #1 whichreacted with horseradish peroxidase-conjugated igG of goat anti-humanIgG Fc in ELISA analyses, which indicated that the constant region ofChi #1 was human. Cytofluorometry analysis indicated that Chi #1 stainedhuman colorectal carcinoma cell lines HT-29 and LS174T but not a humanlung carcinoma cell line A-427. Antibody-dependent cell-mediatedcytoxicity (ADCC) assay indicated that Chi #1 lysed Ls174T cells. Theseresults shows that Chi #1 retained the antigen-binding specificity ofthe parental 31.1 mouse monoclonal antibody, suggesting the useful ofthis chimeric antibody in ascertaining prognosis of colon carcinoma.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

1. A monoclonal antibody specific for a purified human coloncarcinoma-associated protein antigen, wherein said antigen has thefollowing characteristics: (a) said antigen is purified to the extentthat the membrane fractions are free of HL-A antigen and aresubstantially free from non-immunogenic glycoprotein fractions; (b) saidantigen is not detectable on normal colon cancer free human tissues; (c)said antigen is not detectable on human carcinoma cells other than coloncarcinoma cells; (d) said antigen is specifically immunogenic in humans;and (e) said antigen induces an immune response in humans having coloncarcinoma which is expressed as cell mediated immunity which is murinemonoclonal antibody 33.28 as produced by hybridoma PCA 33.28, depositedwith the American Type Culture Collection and assigned accession numberPTA-5413.
 2. An antibody according to claim 1 which is mouse monoclonalantibody 33.28 (ATCC HB-12315) or an antibody which binds specificallyto a colon carcinoma-associated epitope that specifically binds tomonoclonal antibody
 3328. 3. An antibody according to claim 2 1 whereinsaid colon carcinoma-associated antigen is a protein having a molecularweight of about 61.1 kilodaltons as measured by gradient polyacrylamidegel electrophoresis.
 4. An antibody according claim 1 A monoclonalantibody specific for a purified human colon carcinoma-associatedprotein antigen, which is mouse monoclonal antibody 31.1, as produced byhybridoma PCA 31.1 (ATCC HB-12314) , deposited with the American TypeCulture Collection and assigned accession number PTA-2497or an antibodywhich binds specifically to a colon carcinoma-associated epitope thatspecifically binds to monoclonal antibody 31.1 .
 5. An A monoclonalantibody according to claim 4 wherein said colon carcinoma-associatedantigen is a protein having a molecular weight of about 72 kilodaltonswhich comprises an antigen-binding region obtained from the H chain of amurine monoclonal antibody 33.28 as produced by hybridoma PCA 33.28,deposited with the American Type Culture collection and assigned actionnumber PTA-5413.
 6. An antibody according to claim 2 1 wherein saidcolon carcinoma-associated antigen is a glycoprotien, the proteincomponent having a molecular weight of 61.1 kilodaltons as measured bygradient polyacrylamide gel electrophoresis.
 7. An antibody according toclaim 1, 4 or 5 immobilized on a solid phase.
 8. An antibody accordingto claim 1, 4 or 5 which is detectably labeled.
 9. An antibody accordingto claim 8 wherein said detectable label is a radiolabel.
 10. Anantibody according to claim 1, 4 or 5 conjugated to a cytotoxicradionuclide.
 11. An antibody according to claim 1, 4 or 5 conjugated toa cytotoxic drug.
 12. An antibody according to claim 1, 4 or 5conjugated to a cytotoxic protein.
 13. A composition comprising anantibody according to claim 10 in combination with a pharmaceuticallyacceptable carrier.
 14. A composition comprising an antibody accordingto claim 11 in combination with a pharmaceutically acceptable carrier.15. A composition comprising an antibody according to claim 12 incombination with a pharmaceutically acceptable carrier.
 16. A monoclonalantibody against the monoclonal antibody of claim
 1. 17. A monoclonalantibody against the monoclonal antibody of claim
 2. 18. A monoclonalantibody against the monoclonal antibody of claim
 3. 19. A monoclonalantibody against the monoclonal antibody of claim
 4. 20. A monoclonalantibody against the monoclonal antibody of claim
 5. 21. A monoclonalantibody against the monoclonal antibody of claim
 6. 22. An immunoassayfor detecting a colon carcinoma-associated antigen which binds to mousemonoclonal antibody 33.28 (ATCC HB-12315) as produced by hybridoma PCA33.28, deposited with the American Type Culture Collection and assignedaccession number PTA-5413, in a sample comprising: (a) contacting saidsample with an effective binding amount of the antibody according toclaim 1; and (b) detecting said antigen by detecting the binding of theantibody to the purified colon carcinoma-associated protein antigen. 23.An immunoassay for detecting a colon carcinoma-associated antigen whichbinds to mouse monoclonal antibody 31.1 (ATCC HB-12314) , as produced byhybridoma PCA 31.1, deposited with the American Type Culture Collectionand assigned accession number PTA-2497, in a sample comprising: (a)contacting said sample with an effective binding amount of the antibodyaccording to claim 1 4 or claim 5; and (b) detecting said antigen bydetecting the binding of the antibody to the purified coloncarcinoma-associated protein antigen.
 24. A method for diagnosing coloncancer in humans comprising: (a) removing a histological specimen from apatient suspected of having a colon cancer; (b) contacting the specimenwith monoclonal antibody 33.28 (ATCC HB-12315) , as produced byhybridoma PCA 33.28, deposited with the American Type Culture Collectionand assigned accession number PTA-5413; (c) staining the specimen withan immunohistochemical stain; and (d) detecting the presence of theantigen-antibody complex by the stain.
 25. A method for diagnosing coloncancer in humans comprising: (a) removing a histological specimen from apatient suspected of having colon- carcinoma; (b) contacting thespecimen with mouse monoclonal antibody 31.1 (ATCC HB-12314) , asproduced by hybridoma PCA 31.1, deposited with the American Type CultureCollection and assigned accession number PTA-2497); (c) staining thespecimen with an immunohistochemical stain; and (d) detecting thepresence of the antigen-antibody complex.
 26. A method according toclaim 24 wherein the stain is an avidin-biotin immunoperoxidase stain.27. A method according to claim 25 wherein the stain is an avidin-biotinimmunoperoxidase stain.
 28. A kit for the immunohistochemical detectionof colon carcinoma comprising: (a) mouse monoclonal antibody 31.1 (ATCCHB-12314) , as produced by hybridoma PCA 31.1, deposited with theAmerican Type Culture Collection and assigned accession number PTA-2497;(b) reagents for immunoperoxidase and secondary antibody; (c)immunoperoxidase; and (d) colorizing reagents.
 29. A kit for theimmunohistochemical detection of colon carcinoma comprising: (a) mousemonoclonal antibody 33.28 (ATCC HB-12315 , as produced by hybridoma PCA33.28, deposited with the American Type Culture Collection and assignedaccession number PTA-5413; (b) reagents for immunoperoxidase andsecondary antibody; (c) immunoperoxidase; and (d) colorizing reagents.30. A compartmentalized kit for the detection of a human coloncarcinoma-associated antigen, wherein the antigen has the followingcharacteristics: (a) said antigen is purified to the extent that themembrane fractions are free of HL-A antigen and are substantially freefrom non-immunogenic glycoprotein fractions; (b) said antigen is notdetectable on normal colon cancer free human tissues; (c) said antigenis not detectable on human carcinoma cells other than colon carcinomacells; (d) said antigen is specifically immunogenic in humans; and (e)said antigen induces an immune response in humans having colon carcinomawhich is expressed as cell mediated immunity, said kit comprising afirst container adapted to contain an antibody to said antigen or anactive component thereof, and a second container adapted to contain asecond antibody to said antigen or an active component thereof, saidsecond antibody being labeled with a reporter molecule capable of givinga detectable signal.
 31. A kit according to claim 30 wherein thereporter molecule is a radioisotope, an enzyme, a fluorescent molecule,a chemiluminescent molecule or a bioluminescent molecule.
 32. A kitaccording to claim 30 wherein the reporter molecule is an enzyme.
 33. Akit according to claim 30 wherein the kit further comprises a thirdcontainer adapted to contain a substrate for the enzyme.
 34. Acompartmentalized kit for the detection of a human coloncarcinoma-associated antigen, wherein the antigen has the followingcharacteristics: (a) said antigen is purified to the extent that themembrane fractions are free of HL-A antigen and are substantially freefrom non-immunogenic glycoprotein fractions; (b) said antigen is notdetectable on normal colon cancer free human tissues; (c) said antigenis not detectable on human carcinoma cells other than colon carcinomacells; (d) said antigen is specifically immunogenic in humans; and (e)said antigen induces an immune response in humans having colon carcinomawhich is expressed as cell mediated immunity, said kit comprising afirst container adapted to contain monoclonal antibody 31.1 (ATCCHB-12314) to said antigen and a second container adapted to contain asecond antibody to said antigen or an active component thereof, saidsecond antibody being labeled with a reporter molecule capable of givinga detectable signal.
 35. A kit according to claim 34 wherein thereporter molecule is a radioisotope, an enzyme, a fluorescent molecule,a chemiluminescent molecule or a bioluminescent molecule.
 36. A kitaccording to claim 32 wherein the reporter molecule is an enzyme.
 37. Akit according to claim 33 wherein the kit further comprises a thirdcontainer adapted to contain a substrate for the enzyme.
 38. Acompartmentalized kit for the detection of a human coloncarcinoma-associated antigen, wherein the antigen has the followingcharacteristics: (a) said antigen is purified to the extent that themembrane fractions are free of HL-A antigen and are substantially freefrom non-immunogenic glycoprotein fractions; (b) said antigen is notdetectable on normal colon cancer free human tissues; (c) said antigenis not detectable on human carcinoma cells other than colon carcinomacells; (d) said antigen is specifically immunogenic in humans; and (e)said antigen induces an immune response in humans having colon carcinomawhich is expressed as cell mediated immunity, said kit comprising afirst container adapted to contain monoclonal antibody 33.28 (ATCCHB-12315) to said antigen and a second container adapted to contain asecond antibody to said antigen or an active component thereof, saidsecond antibody being labeled with a reporter molecule capable of givinga detectable signal.
 39. A kit according to claim 38 wherein thereporter molecule is a radioisotope, an enzyme, a fluorescent molecule,a chemiluminescent molecule or a bioluminescent molecule.
 40. A kitaccording to claim 38 wherein the reporter molecule is an enzyme.
 41. Akit according to claim 38 wherein the kit further comprises a thirdcontainer adapted to contain a substrate for the enzyme.
 42. Themonoclonal antibody of claim 1 which is a chimeric antibody.
 43. The Achimeric antibody according to claim 42 which is a chimeric mouse/humanantibody Chi #1 as produced by the cell line deposited with the AmericanType Culture Collection and assigned accession number (ATCC CRL-12316) .44. The chimeric antibody according to claim 42 wherein said coloncarcinoma-associated antigen is a protein having a molecular weight of72 kilodalton.
 45. A composition comprising the chimeric antibodyaccording to claim 42 43 in combination with a pharmaceuticallyacceptable carrier.
 46. A monoclonal antibody against the chimericantibody of claim
 42. 47. An immunoassay for detecting a coloncarcinoma-associated antigen which binds to the mouse/human chimericantibody Chi #1 as produced by the cell line deposited with the AmericanType Culture Collection and assigned accession number (ATCC CRL-12316)of claim 42 in a sample comprising: (a) contacting said sample with theChi #1 antibody according to claim 42 ; and (b) detecting said antigenby detecting the binding of said antibody to the purified coloncarcinoma-associated protein antigen.
 48. A method for diagnosing coloncancer in humans comprising: (a) removing a histological specimen from apatient suspected of having a colon carcinoma; (b) contacting thespecimen with a chimeric antibody which binds to an antigen according toclaim 1 43; (c) staining the specimen with an immunohistochemical stain;and (d) detecting the presence of the antigen-antibody complex by thestain.
 49. A method for diagnosing colon cancer in humans comprising:(a) removing a histological specimen from a patient suspected of havinga colon carcinoma; (b) contacting the specimen with mouse/human chimericantibody which binds to an antigen which binds to mouse/human chimericantibody Chi #1 (ATCC as produced by the cell line deposited with theAmerican Type Culture Collection and assigned accession numberCRL-12316) ; (c) staining the specimen with an immunohistochemcialstain; and (d) detecting the presence of the antigen-antibody complex bythe stain.
 50. A kit for the immunohistochemical detection of coloncarcinoma comprising: (a) mouse/human chimeric antibody Chi #1 (ATCCCRL-12316); (b) reagents for immunoperoxidase and secondary antibody;(c) immunoperoxidase; and (d) colorizing reagents.